Microtubules represent one of the three essential cytoskeleton types in cells. Important for a variety of physiological functions, encompassing cell migration, mitosis, neuronal differentiation and transport of cargo, microtubule-associated motor proteins have been implicated in numerous diseases, ranging from motor neuron and degenerative disorders, to neoplasia and viral infections. Microtubule-binding CAP-Gly domains are conserved in organisms from human to yeast, play central roles in many proteins, and their mutations lead to various disorders. CAP-Gly domain of the p150glued subunit of dynactin interacts with microtubules, and its mutations are associated with several motor neuron disorders. The atomic-level structure and dynamics of CAP-Gly/microtubule assemblies are not known because of their inherent insolubility and lack of long-range order. Lack of such insight hampers further research and impedes design of effective therapies against diseases associated with cytoskeleton dysfunction. Our long-term goal is to understand the structural and dynamic basis of cargo transport regulation along microtubules by microtubule-associated proteins, in healthy and disease states. The objectives of this application are to determine three-dimensional structures and dynamics of CAP-Gly domain of dynactin and of its macromolecular assemblies with the microtubules and with EB1 protein. We will employ multidimensional high-resolution magic angle spinning solid-state NMR methods in conjunction with biophysical and biochemical techniques. In the specific aims designed to accomplish the objectives of this application, we will: 1) determine the structure of CAP-Gly alone and CAP-Gly assembled on the microtubule, and identify the CAP-Gly/microtubule interface at atomic resolution; 2) characterize the energetics and dynamics of the CAP-Gly/microtubule interaction; 3) characterize the dynamics of CAP-Gly mutants related to neurological pathologies; 4) characterize biochemically and structurally the regulation of the CAP- Gly/EB1/microtubule interaction. The proposed work has important implications for human health as it will shed light on the structure of CAP-Gly:microtubule complexes that are not amenable to structural characterization by X-ray crystallography or solution NMR spectroscopy, and will enable structural characterization of macromolecular assemblies consisting of microtubule-associated proteins in complexes with microtubules. PUBLIC HEALTH RELEVANCE: Microtubules represent one of the three essential types of cytoskeleton in cells and, together with their associated proteins, play important roles in a broad range of physiological functions, encompassing cell migration, mitosis, polarization and differentiation, and vesicle and organelle transport. Microtubule-associated proteins have been implicated in numerous diseases ranging from motor neuron and degenerative disorders, to neoplasia and viral infections. Atomic-resolution structures and dynamics of microtubule assemblies with their associated proteins are not known due to their intrinsic insolubility and lack of long range order. Lack of such insight hampers further research and impedes design of effective therapies against diseases associated with cytoskeleton dysfunction. The research proposed in this application will fill this knowledge gap by providing the atomic-resolution structure and dynamics of the microtubule-associated CAP-Gly domain of the p150Glued subunit of dynactin bound to the microtubules. State-of-the-art solid-state NMR spectroscopy will be introduced as a novel technique to probe the intrinsically insoluble and non-crystalline assemblies of microtubules with their associated proteins.